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HOW STEM CELLS AND GENE THERAPY MIGHT WORK TOGETHER

A sample of bone marrow is removed. Stem cells are isolated and allowed to multiply in culture. Cells are treated with a modified virus containing a therapeutic gene. The virus is taken up by individual cells and the therapeutic gene goes into the cell's nucleus. Treated ("corrected") cells are injected into the bloodstream. Treated cells respond to injury signals from degenerating muscle or other tissues and migrate out of the bloodstream. Treated cells patch damage and build healthy tissue.

Stem Cell Applications

If we can learn to harness the potential of stem cells, the applications are endless. Already researchers are talking about converting stem cells to insulin-producing cells for transplant into the pancreases of people with diabetes. Others speculate that whole organs might one day be cultured in the lab from a person's own stem cells to make immuno-compatible replacement parts. Others talk of injecting stem cells into the blood or spinal fluid and allowing them to make their own way in the body to repair distributed tissues like muscle and nerve. If the recipient of the stem cells happens to have a genetic disease, the cells could be obtained from a healthy donor, or be treated with gene therapy to correct the mutation before being reinserted in the recipient (see illustration, at left).

If successful, stem cell transplants could eventually be used to produce new, healthy muscle in people with almost any kind of genetic muscle disorder, including the congenital, metabolic and mitochondrial myo-pathies. Meanwhile, stem cells in the nervous system might reconnect the brain to the muscles in people with spinal muscular atrophy or amyotrophic lateral sclerosis, or speed the transmission of impulses in people with Charcot-Marie-Tooth disease.

Progress in Making New Muscle

For MDA grantee Louis Kunkel of Children's Hospital in Boston, who first identified the gene that's defective in Duchenne muscular dystrophy, cell transplantation has been a long-standing interest for the treatment of neuromuscular disease.

Kunkel followed closely the early work in myoblast transfer, but was disappointed in the low efficiency associated with injecting and maintaining these immature cells in muscle. By the summer of 1998, Kunkel and his collaborator, Richard Mulligan of Harvard Medical School in Boston, were wondering if they could purify more useful cells from muscle by modifying a new technique for sorting stem cells from bone marrow.

Their approach was successful. The researchers managed to separate cells from muscle tissue that had some of the same characteristics of bone marrow stem cells. They were preparing to inject healthy cells isolated by the new method into the muscles of mice with a faulty gene, when work by Italian researchers describing how a bone marrow transplant could contribute to muscle caught their attention. They decided instead to test their muscle cells in mice in the same way that a bone marrow transplant is performed - by first destroying the host bone marrow through irradiation, then injecting the stem cells directly into the bloodstream.

To their amazement, the transplanted muscle stem cells not only regenerated the recipient's bone marrow, but also contributed to the recipient mouse's voluntary muscle. And, because the stem cells were originally isolated from a healthy mouse, the new muscle cells had fully functional genes for dystrophin, the protein missing in Duchenne dystrophy.

"Essentially, we had fixed, through injection into the bloodstream, all of those muscle fibers that we got these cells into," Kunkel says. "What's amazing is that they contributed enormously, up to almost 9 percent at four weeks, to the remodeling of the muscle."

Emanuela Gussoni, a postdoctoral associate in Kunkel's lab, says researchers suspect the muscle stem cells isolated in the new manner are a more primitive form of myoblasts (immature muscle cells) that have the potential to contribute to multiple tissues. However, the exact difference, if any, between the two populations of cells isn't yet known.

Kunkel and Mulligan also found that stem cells isolated from the bone marrow, rather than the muscle, of a healthy donor could give rise to dystrophin-positive muscle fibers.

"So we've now got a cell that has the capability of remodeling muscle from the circulation of the organism and delivering gene product [dystrophin] to that remodeled muscle," Kunkel says. "What this work is really doing is opening a whole field of expanding stem cell biology as a potential treatment for genetic disorders, and specifically for Duchenne and the other muscular dystrophies."

Although the level of dystrophin-positive fibers resulting from these initial transplants isn't high enough to restore muscle function in mice, researchers are working on ways to increase the number of positive fibers and stimulate stem cell migration into the muscle, without having to destroy the bone marrow of the recipient.

"Here's where you have to be cautious," Kunkel says. "We are not going to irradiate children with DMD. What we want to do is find the signal from muscle that says 'I am injured, come help me' to the circulating donor stem cells. Then we can use that signal in some nontoxic way to lure donor stem cells to the muscle."

The Italian researchers' article also caught the eye of Margaret Goodell of Baylor College of Medicine in Houston, whose laboratory studies the stem cells that produce blood. Goodell had developed the new method for sorting bone marrow stem cells that was modified by Kunkel and Mulligan to extract stem cells from muscle.

When Goodell applied her unmodified stem cell purification procedure to muscle, she isolated muscle-derived stem cells that were even better than bone marrow stem cells at regenerating the bone marrow of host mice.

Goodell is also interested in doing the reverse: regenerating muscle in people with genetic muscle diseases through a sort of bone marrow transplant.

"I think one of the most significant things about stem cells is that they provide a new partner to gene therapy - a sort of 'cell therapy.' Ultimately, the best option would be to take someone's own stem cells from bone marrow or muscle and do gene therapy to correct the problem, and then transplant them," she says.

For now, however, Goodell says there are some problems with doing gene therapy in stem cells, so getting healthy cells from an immunologically matched donor is a more viable option. Both Kunkel and Goodell agree that more work needs to be done on the basic biology of adult-derived stem cells, especially in understanding the signals that draw the stem cells out of the circulation to sites of injury.

Rewiring the Nervous System

One of the most exciting potential applications for stem cells is regrowing lost human nerve cells - a feat first hinted at more than a decade ago when researchers learned how to isolate stem cells from a mouse brain, grow them in the lab, treat them with therapeutic genes, then return them to mouse brains where they became normal brain cells.

This type of strategy for humans wasn't considered seriously until about two years ago, when researchers found that human neural stem cells could be isolated, treated and reimplanted into the brains of mice to replace missing cells.

They also found that the human brain, just like the rodent brain, has an area where new neurons continue to be produced well into old age. The latter finding suggests that the replacement of brain cells in humans may be a normal process that could be augmented by stem cells. Evan Snyder of Children's Hospital in Boston was the first to isolate human neural stem cells. However, his most dramatic finding occurred when he demonstrated soon after that transplanted neural stem cells could correct "global" degenerative conditions that are distributed throughout the brain.

To do this, he transplanted healthy donor neural stem cells into the brains of newborn mice that had a genetic disease that caused them to shake constantly because of a depletion of the brain's insulating material, myelin.

By one to two weeks after transplant, the healthy donor cells had migrated throughout the brains of the recipient mice and had transformed into the type of nerve cell that makes myelin. Some animals even experienced a decrease in the amount of tremor.

These findings bode well for replacing cells that are distributed throughout different regions of the brain, but more work needs to be done to make the technique practical to treat conditions involving motor neurons, the nerve cells that are affected in ALS and SMA. The first problem is making sure that the stem cells can be delivered efficiently and reproducibly to all the necessary areas, Snyder says.

"For instance, in a disease like ALS or SMA, where it's not just one segment that's involved, but many segments, this would be necessary," he explains. He says the second obstacle is making sure that the transplanted stem cells not only become motor neurons, but that they send out their connections in the right directions and get hooked up properly.

But the research has come a long way, Snyder says.

He has preliminary results showing that mouse neural stem cells can become motor neurons when transplanted into a mouse in which the motor neurons have been triggered to degenerate through injury. He also has early evidence that human neural stem cells might be able to become "at least a few motor neurons."

Still, one practicality in trying to replace motor neurons is that many of these cells have unusually long extensions, up to 3 feet for the motor neurons that control the muscles of the big toe.

Snyder says that the regrowth process could probably be accelerated with the right types of growth factors and points out that some target muscles are closer than others. A good example is the bulbar muscles, which are involved in swallowing and are frequently affected in ALS.

Overall, Snyder is very enthusiastic about the future of neural stem cell-based treatments. "This is a pretty exciting period of time for us," he says. "I think we're going to make a major impact over the next decade, or certainly the next quarter century on what happens with nervous system diseases."

Staying on Top of the Science

As progress in stem cell research gains momentum, MDA has launched a major research initiative in this area. Last summer, some of the best minds in this field, including the researchers mentioned in this article, and many others, were invited to a special MDA meeting to discuss applications of stem cell biology in the treatment of neuromuscular disease.

Much original research was presented and topics were discussed informally. In the end, many members of the group were astounded to find that the stem cells they were studying from different tissues all had very similar characteristics. As a direct result of this meeting, several collaborations were initiated and research proposals came flooding in.

Now a second, much larger meeting, the Orthopeaedic and Tissue Engineering Symposium, co-sponsored by MDA, is scheduled in April in Pittsburgh. Many of the sessions address therapies for neuromuscular disease.